1. Field of the Invention
The present invention relates to novel expression vectors and a method to increase the expression of .alpha..sub.1 -antitrypsin (AAT) from expression vectors encoding the same through the inclusion of the Intron II sequence.
2. Background of the Art
Alpha.sub.1 -antitrypsin (AAT) deficiency is one of the most common inherited disorders in the US, affecting an estimated 20,000-40,000 individuals. AAT is a relatively small plasma glycoprotein with 394 amino acids and three oligosaccharide sidechains. AAT is a member of the serine protease inhibitor superfamily. The serine protease inhibitor family consists of at least 12 genes, most of which are involved in the control of serine proteases in blood coagulation, in complement activation, and in certain aspects of inflammation reactions. The family members are believed to have evolved from a common ancestor gene over about 500 million years.
The gene that encodes AAT in the liver has been identified as a 10.2 kb gene, consisting of five exons, four of which together encode that protein, and four introns. Perlino et al. EMBO J. 6:2767-2771 (1987). The exons are separated by a series of introns or intervening sequences.
There are two categories of AAT defects that cause disease states. The first category includes the deficient allele, in which AAT is present in low levels in the blood serum. The second category is the null alleles, in which no AAT in the blood serum can be detected.
AAT is synthesized primarily in the liver and subsequently secreted into the bloodstream where it serves as the predominant serine protease inhibitor. Although AAT is capable of inhibiting a variety of proteases including trypsin, chymotrypsin, thrombin, kallikrein, and plasmin (Laurell et al. The Plasma Proteins Vol 1, pp. 229-264 (Academic Press, New York, (F. W. Putnam, Ed. 1975)), its major physiological role is to protect elastic tissues in the alveoli structure of the lung from hydrolysis by excessive neutrophil elastase.
Clinically, AAT is important in that its genetic deficiency predisposes individuals toward the development of pulmonary emphysema. However, it is also an important genetic disease in its manner of manifestation. In children, the deficiency is manifested as liver disease and leads to the second leading reason for liver transplants in children. In adults, the deficiency is manifested primarily in the lungs, with secondary manifestations in the liver. In the lungs, lack of AAT antiprotease activity from AAT deficiency results in the uncontrolled breakdown of alveolar connective tissue leading to emphysema (Gadek et al. The Metabolic Basis of Inherited Disease pp. 1450-1467 (5th edition, McGraw-Hill, New York (1982)). For this reason, inheritance of two deficient AAT alleles substantially increases an individual's risk of developing emphysema or liver disease.
Accordingly, many investigators have been studying the gene regulation of AAT in an attempt to determine methods of treating individuals having the gene deficiency.
For example, Long et al. Fed. Proc. Fed. Am. Soc. Exp. Biol. 42(7):1761 (1983) disclosed the DNA sequence of the .alpha.1-antitrypsin gene and studied the structure of a large intron corresponding to the 5' untranslated portion of the mRNA. Matsunaga et al. Am. J. Hum. Genet. 46(3):602-612 (1990) disclosed the sequence of .alpha..sub.1 -antitrypsin mutants as compared with normal .alpha..sub.1 -antitrypsin gene sequences. Their results revealed that there were differences between the intron sequences of the mutant and wild-type genes, although these differences did not result in different gene splicing patterns.
PCT Publication No. 90/05188 to Archibald et al. disclosed a method for producing large amounts of a medically important human proteins in the milk of transgenic animals by producing a construct encoding the medically important protein that contained an intron from that same protein. Archibald et al. incorporated DNA encoding for a heterologous protein together with at least one intron into a fusion protein that is a mammalian milk protein gene. The application indicated that, advantageously, high levels of protein expression were obtained from constructs employing some, but not all, naturally occurring introns in a gene.
Archibald et al. used .alpha..sub.1 -antitrypsin as one of its examples. In addition, the Archibald et al. application also cites a paper (Brinster et al., Proc. Natl. Acad. Sci. (USA) 5:836-840 (1988)), in which increased transcriptional efficiency is reportedly achieved by incorporating introns into transgenes in transgenic mice and that, importantly, introns from the native genome sequences yielded more efficient gene expression than foreign introns. However, Brinster et al. indicated that the effect is unique to transgenic animals and was not observed in cell lines. The Archibald et al. application goes on to cite the problems associated with manipulating large genome sequences containing all of the introns associated with a gene.
Others have also looked at introns as means for increasing gene expression. U.S. Pat. No. 5,108,909 to Haigwood discloses a method for improving expression of tissue plasminogen activator (tPA) in a mammalian cell by incorporating at least one tPA associated intron into an expression construct operably encoding tPA, where the intron is positioned in its native location.
Reid et al. Proc. Natl. Acad. Sci. (USA) 87(11):4299-4303 (1990) disclosed that hypoxanthine phosphoribosyltransferase (HPRT) required one HPRT intron for efficient cell expression. The intron's presence was not required for splicing and was not associated with a traditional transcription enhancer element that had been identified in another HPRT intron. While Brintner et al. (supra) limited the observation to transgenic animals, Reid et al., demonstrated the effect in somatic cells.
Jonsson et al. Nucl. Acids Res. 20(12):3191-3198 (1992) disclosed the addition of the first intron of purine-nucleoside orthophosphate ribosyltransferase (PNP) into a construct for PNP gene expression. They disclose the use of these PNP minigenes for retroviral-mediated gene transfer. Similarly, Chan et al. Gene 73(2):295-304 (1988) disclosed the use of E. coli intron sequences to increase the stability of chloramphenicol acetyl transferase (CAT) in bacteria.
Choi et al. Mol. Cell. Biol. 11(6):3070-74 (1991) disclosed using a heterologous intron consisting of an adenovirus splice donor and an immunoglobulin G splice acceptor to stimulate expression of CAT in a variety of tissues in a transgenic animal. Maas et al. Plant. Mol. Biol. 16(2):199-207 (1991) and Mascarenhas et al. Plant Mol. Biol. 15(6):913-920 (1990) disclosed the enhanced expression of CAT in maize protoplasts by including introns from the maize alcohol dehydrogenase gene.
The above variety of intron effects on genomic expression of mRNA and protein demonstrate that the mode of action of introns in expression is not well understood. It is not until one or more introns are inserted and others are excluded from a vector and levels of expression measured, can one predict the effect that may be caused by the presence or absence of a particular intron.
Many disease states, such as those discussed above, might be treated by introduction of a gene encoding Alpha1-antitrypsin. Unfortunately, cloning of the complete gene is very difficult in retroviral or adenoviral shuttle vectors because of limitations in the size of insert DNA. In addition, gene expression with the .alpha.1-antitrypsin cDNA has been found to be too low for use as a mechanism of treatment, regardless of the promoter used. It would provide a great advantage to have a gene that was capable of expressing high levels of AAT protein, but preferably, still be small enough to fit in a retroviral or adenoviral shuttle vector, i.e., less than or equal to 3000 bp in length.